Targeting g3bp proteins to accelerate nerve regeneration

ABSTRACT

Methods for using a peptide to effectively increase axon growth in both naive and injury-conditioned neurons.

This invention was made with government support under W81XWH-2013-1-308;OR120042 awarded by the Department of Defense and R01-NS041596 awardedby the National Institute of Health. The government has certain rightsin the invention.

BACKGROUND OF THE INVENTION 1) Field of the Invention

The present invention relates to vehicles and methods for improvingnerve regeneration after nerve injury.

2) Description of Related Art

“Peripheral nerve” is a term used synonymously to describe theperipheral nervous system. The peripheral nervous system is a network ofmotor and sensory nerves that connect the brain and spinal cord (thecentral nervous system or CNS) to the entire human body. These nervescontrol the functions of sensation, movement and motor coordination. Theperipheral nerves are a complicated, extensive network of nerves thatare the tool for the brain and spinal cord to communicate with the restof the body. They are fragile and can be damaged easily. Trauma,including battlefield injuries, is a major cause of peripheral nerveinjury along with birth trauma (brachial plexus injury) and injuriesrelated to surgeries. Limited data is available to determine theincidence of peripheral nerve injury.

Neurons, nerve cells that are the basic building block of the nervoussystem, generate their own proteins within cytoplasmic processes thatextend for centimeters in rodents and more than a meter in humans. Theselocally generated proteins are needed for regeneration after nerveinjury. Protein synthesis in axons contributes to axon growth duringdevelopment and regeneration.

The stress response in eukaryotic cells often inhibits translationinitiation and leads to the formation of cytoplasmic RNA-proteincomplexes referred to as stress granules. Stress granules, denseaggregations in the cytosol (the aqueous component of the cytoplasm of acell) comprised of proteins and RNAs, are used to store mRNAs duringperiods of cellular stress, but nerve injury with compromise of axonintegrity paradoxically causes stress granules to disaggregatepresumably releasing mRNAs for translation. Stress granules containnon-translating mRNAs, translation initiation components, and manyadditional proteins affecting mRNA function. Stress granules have beenproposed to affect mRNA translation and stability, as well as beinglinked to apoptosis and nuclear processes. Stress granules also interactwith P-bodies, another cytoplasmic RNP granule containingnon-translating mRNA, translation repressors and some mRNA degradationmachinery. Together, stress granules and P-bodies reveal a dynamic cycleof distinct biochemical and mRNA-protein complexes (mRNPs) in thecytosol, with implications for the control of mRNA function.

Nerve regeneration is abysmally slow in the peripheral nervous systemand does not occur spontaneously in the central nervous system. Despitethat regeneration occurs in the periphery, the slow growth in humansmeans that by the time regeneration occurs the distal nerve is no longera growth-supportive environment and the target tissues are no longerreceptive for reinnervation that restores the nerve supply to a part ofthe body. There is a pressing clinical need for treatments that willaccelerate axon regeneration in peripheral nerves.

Regeneration in the brain and spinal cord is thought to fail because ofextrinsic inhibitors of axon growth and the low intrinsic growthpotential of central nervous system neurons. Some approaches to increaseintrinsic growth potential of neurons have been shown to also overcomethe extrinsic growth inhibitors. There is a pressing clinical need forbetter agents to increase intrinsic growth that directly target theaxons.

Current products in use clinically consist of a myriad of ‘gap bridging’approaches for peripheral nerve treatments. Experimentally, these havebeen encapsulated with various growth promoting agents (e.g., growthfactors), but none have specifically targeted regeneration rates. Otherapproaches include exercise, which is used clinically forrehabilitation/physical therapy, but likely has some regenerativeeffects. For both stroke and spinal cord injury, rehabilitation issimilarly used in clinics. Experimental evidence points to some enhancedregeneration with these techniques.

Accordingly, it is an object of the present invention to providevehicles and methods for improving nerve regeneration after nerveinjury.

SUMMARY OF THE INVENTION

In a first embodiment, a method for treating nerve injury in a mammal isprovided. The method may include introducing a polypeptide comprisingbetween 15 and 20 amino acids to a nerve injury site in the mammal. Thepolypeptide may interfere with function of stress granules and increasesintra-axonal rates of translation of proteins needed for nerveregeneration. Further, the polypeptide may be an amino acid sequence setas forth in SEQ ID NO. 2. Still further, the polypeptide specificallymay target mRNA storage sites in neurons and increases rates of neuronregeneration. Even further, the polypeptide may disrupt G3BP function.Further yet, disruption of G3BP functions may include activatingintra-axonal mRNA translation, increasing axon growth in neurons, andaccelerating nerve regeneration in vivo. Furthermore, disruption of G3BPfunctions may be accomplished via siRNA-mediated knockdown of G3BP1.Further yet still, disrupting G3BP1's function in an assembly of axonalstress granule structures may increase intra-axonal protein synthesisand accelerate peripheral nervous system axon regeneration. Even stillfurther, accelerated axon growth regeneration may be facilitated bysequestering Imp81 mRNA from translation.

In an alternative embodiment, a method of disrupting G3BP functions isprovided. The method may include overexpressing a dominant-negativeprotein. The dominant-negative protein may: disassemble axonal stressgranule-like structures, activate intra-axonal mRNA translation,increase axon growth in neurons; and accelerate nerve regeneration invivo. Further, the protein may comprise between 15 and 20 amino acids.Still yet, the protein may have an amino acid sequence as set forth inSEQ ID NO. 2. Further still, the protein may be cell permeable andtarget mRNA storage sites in neurons. Yet further, disruption of G3BPfunctions may be accomplished via siRNA-mediated knockdown of G3BP1.Further yet still, disrupting G3BP1's function in an assembly of axonalstress granule structures may increase intra-axonal protein synthesisand accelerate peripheral nervous system axon regeneration. Stillfurthermore, preventing stress granule-like aggregation of axonalproteins during regeneration may increase the rate of axon regrowth.

BRIEF DESCRIPTION OF THE DRAWINGS

The construction designed to carry out the invention will hereinafter bedescribed, together with other features thereof. The invention will bemore readily understood from a reading of the following specificationand by reference to the accompanying drawings forming a part thereof,wherein an example of the invention is shown and wherein:

FIG. 1 shows a schematic of how translation is regulated in axons.

FIG. 2 shows an immunofluorescence comparison of the presence of G3BP1protein in axons pre-injury and post-injury.

FIG. 3 shows an immunofluorescence (IF) comparison and a graphillustrating the differences in phosphorylated G3BP in axons.

FIG. 4 shows an immunofluorescence comparison and a graph ofdifferential colocalization of G3BP with axonal mRNAs in axons of naivevs. injury-conditioned neurons.

FIG. 5 shows an immunofluorescence comparison and a graph showingexpression of known G3BP domains vis-A-vis axon growth.

FIG. 6 shows an immunofluorescence comparison and a graph illustratingthat G3BP B-domain increases axon regeneration in vivo.

FIG. 7 shows a graph illustrating that the peptide for G3BP1 residuessupports axon growth in both PNS and CNS neurons as well as a predicted3D structure of the peptide.

FIG. 8A shows Immunofluorescence for G3BP1 shows signals in cell body(asterisk) and along distal neurites (arrows) in a cultured DRG neuron.

FIG. 8B shows single optical planes for axons of naïve DRG cultures.

FIG. 8C also shows single optical planes for axons of naïve DRGcultures.

FIG. 8D shows exposure-matched images of G3BP1 in distal axons of DRGscultured from naïve vs. 7 day injury conditioned animals.

FIG. 8E shows quantification of axonal G3BP1 immunoreactivity fromsciatic nerve.

FIG. 8F shows quantification of axonal G3BP1 immunoreactivity from axonsof cultured DRG neurons.

FIG. 9A shows axons of DRG neurons transfected with indicated G3BP1constructs vs. eGFP.

FIG. 9B shows quantification of axonal aggregates for G3BP1-GFP,G3BP1^(S149A)-GFP, and G3BP1^(S149E)-GFP.

FIG. 9C shows Flourescence Recovery After Photobleaching (FRAP) analysesfor neurons transfected with constructs from FIG. 9B.

FIG. 9D shows exposure-matched confocal images for G3BP1^(PS149) andneurofilament (NF) for sciatic nerve as in FIG. 8D.

FIG. 9E shows quantification of the signals of FIG. 9D.

FIG. 9F shows distal axons of cultured DRGs immunostained with pan-G3BP1vs. G3BP1^(PS149) antibodies.

FIG. 9G shows quantification of the signals of FIG. 9F.

FIG. 10A shows images of Fluorescence in situ Hybridization (FISH) plusIF for indicated mRNAs and G3BP1 protein for axons of naïve and 7 dinjury-conditioned DRG neurons.

FIG. 10B shows quantification of colocalizations for Nrn1, Impβ1, andGap43 mRNAs with G3BP1 in axons of neurons cultured from naïve or 7 dayinjury-conditioned animals.

FIG. 10C shows schematics of translation reporter constructs used inpanels d-f.

FIG. 10D shows representative FRAP image sequences for DRG neuronsco-transfected with GFP^(MYR)5′/3′nrn1 plus BFP or G3BP1-BFP.

FIG. 10E shows quantifications of FRAP assays from DRGs expressingGFP^(MYR)5′/3′nrn1.

FIG. 10F shows quantifications of FRAP assays from DRGs expressingGFP^(MYR)5′/3′impβ1.

FIG. 10G shows HEK293T cells transfected with GFP^(MYR)5′/3′nrn1,GFP^(MYR)5′/3′ impβ1 and mCh^(MYR)5′/3′gap43.

FIG. 11A shows a schematic of G3BP1 domains as defined by Tourriere etal. (2003).

FIG. 11B shows representative images for NF-labeled DRG neuronstransfected with indicated constructs.

FIG. 11C shows quantitation of axon growth from DRGs (left) and corticalneurons (right) treated with cell-permeable 168-189 or 190-208 G3BP1peptides is shown.

FIG. 11D shows extent of axon regeneration at 7 d post sciatic nervecrush in adult rats transduced with AAV5 encoding G3BP1-BFP, G3BP1 Bdomain-BFP, G3BP1 D domain-BFP, or GFP control.

FIG. 12A show representative images for puromycin (Puro) incorporationin DRG neurons transfected with indicated constructs. Quantitation showsno change in cell body Puro signals (b), while the axons show asignificant increase in Puro signals in the G3BP1 B domain expressingneurons (c) (N≥100 axons and N≥30 cell bodies over 3 repetitions;**p<0.01 by one-way ANOVA with Tukey HSD posthoc) [scale bar=5 μm].

FIG. 12B shows quantitation that shows no change in cell body Purosignals.

FIG. 12C shows that the axons show a significant increase in Purosignals in the G3BP1 B domain expressing neurons (c).

FIG. 12D show puro incorporation in DRG axons was also significantlyincreased by the 190-208 G3BP1 peptide treatment compared to control and168-189 peptide exposure.

FIG. 12E shows RTddPCR for axonal mRNAs associated with G3BP1-GFP in DRGneurons as average % mRNA associated with G3BP1-GFP±SEM.

FIG. 12F show representative images of G3BP1-mCh in DRG axons treatmentwith 190-208 G3BP1 peptide for 15 min.

FIG. 12G show density of G3BP1-mCh aggregates along 100 μm length axonsfrom DRG cultures treated as in FIG. 12F.

FIG. 12H shows the size of G3BP1-mCh aggregate as indicated bins forfrom DRG cultures treated as in FIG. 12F.

FIG. 13A shows representative FRAP image sequences.

FIG. 14A shows FRAP analyses for DRGs expressing mCh^(MYR)5′/3′gap43plus the indicated G3BP1-BFP constructs or BFP control.

FIG. 14B shows FRAP analyses for DRGs expressing GFP^(MYR)5′/3′nrn1 ormGFP^(MYR)5′/3′impβ1 plus the G3BP1 B domain-BFP or G3BP1 D domain-BFP.

FIG. 14C shows FRAP analyses for DRGs expressing GFP^(MYR)5′/3′nrn1,GFP^(MYR)5′/3′impβ1, or mCh^(MYR)5′/3′gap43 plus G3BP1-BFP.

FIG. 15A shows axon growth parameters for DRG neurons transfected as inFIG. 11B.

FIG. 15B shows RT-ddPCR analyses of G3BP1 and G3BP2 mRNA levels in DRGstransfected with control (siCntl) and G3BP1 (siG3BP1) siRNAs.

FIG. 15C shows axon growth data for DRG neurons transfected with control(siCntl) or G3BP1 (siG3BP1) siRNAs plus GFP, G3BP1-GFP, or G3BP1 Bdomain-GFP.

FIG. 16A shows Representative images of dansyl chloride fluorescence ofinternalized peptides in DRG cell bodies.

FIG. 16B shows neurite outgrowth analyses for dissociated DRG neuronstreated with peptides immediately after plating.

FIG. 16C shows representative images of axonal compartment ofmicrofluidic culture device with cortical neurons stained for tau at DIV6.

FIG. 17A shows representative images of distal axons of naïve DRGneurons were transfected with G3BP1 B domain-GFP, D domain-GFP, CDdomain-GFP, or BCD domain-GFP.

FIG. 17B shows representative, exposure-matched confocal images ofcrushed sciatic nerves of adult rats transduced with AAV5 encoding theG3BP1-BFP, G3BP1 B domain-BFP, G3BP1 D domain-BFP or GFP.

FIG. 18A shows representative images for puromycin (Puro) incorporationin DRG neurons treated with indicated peptides.

FIG. 18B shows representative images of HuR immunoreactivity in NIH-3T3cells that were transfected GFP vs. G3BP1 B domain-GFP are shown after30 min treatment with sodium arsenite (0.5 mM).

FIG. 18C shows quantification SGs in the transfected NIH-3T3 cells fromFIG. 18A are shown based on the indicated immunostaining.

FIG. 19 shows one embodiment of a method of the current disclosure.

FIG. 20 shows one embodiment of an alternative method of the currentdisclosure.

It will be understood by those skilled in the art that one or moreaspects of this invention can meet certain objectives, while one or moreother aspects can meet certain other objectives. Each objective may notapply equally, in all its respects, to every aspect of this invention.As such, the preceding objects can be viewed in the alternative withrespect to any one aspect of this invention. These and other objects andfeatures of the invention will become more fully apparent when thefollowing detailed description is read in conjunction with theaccompanying figures and examples. However, it is to be understood thatboth the foregoing summary of the invention and the following detaileddescription are of a preferred embodiment and not restrictive of theinvention or other alternate embodiments of the invention. Inparticular, while the invention is described herein with reference to anumber of specific embodiments, it will be appreciated that thedescription is illustrative of the invention and is not constructed aslimiting of the invention. Various modifications and applications mayoccur to those who are skilled in the art, without departing from thespirit and the scope of the invention, as described by the appendedclaims. Likewise, other objects, features, benefits and advantages ofthe present invention will be apparent from this summary and certainembodiments described below, and will be readily apparent to thoseskilled in the art. Such objects, features, benefits and advantages willbe apparent from the above in conjunction with the accompanyingexamples, data, figures and all reasonable inferences to be drawntherefrom, alone or with consideration of the references incorporatedherein.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

With reference to the drawings, the invention will now be described inmore detail. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood to one ofordinary skill in the art to which the presently disclosed subjectmatter belongs. Although any methods, devices, and materials similar orequivalent to those described herein can be used in the practice ortesting of the presently disclosed subject matter, representativemethods, devices, and materials are herein described.

Unless specifically stated, terms and phrases used in this document, andvariations thereof, unless otherwise expressly stated, should beconstrued as open ended as opposed to limiting. Likewise, a group ofitems linked with the conjunction “and” should not be read as requiringthat each and every one of those items be present in the grouping, butrather should be read as “and/or” unless expressly stated otherwise.Similarly, a group of items linked with the conjunction “or” should notbe read as requiring mutual exclusivity among that group, but rathershould also be read as “and/or” unless expressly stated otherwise.

Furthermore, although items, elements or components of the disclosuremay be described or claimed in the singular, the plural is contemplatedto be within the scope thereof unless limitation to the singular isexplicitly stated. The presence of broadening words and phrases such as“one or more,” “at least,” “but not limited to” or other like phrases insome instances shall not be read to mean that the narrower case isintended or required in instances where such broadening phrases may beabsent.

As there are no currently available treatments to accelerate nerve‘regeneration’. The approach used by the current disclosure targetsprotein synthesis in the distal axon, the compartment of the neuron thatneeds to regenerate to restore function. Consequently, this provides aunique way to increase intrinsic growth potential of the neuron.

The current disclosure presents data on the general mechanism fortargeting the G3BP proteins and efficacy of viral targeting foraccelerating axon regeneration. In one embodiment, a cell permeableG3BP1 peptide is disclosed that may have potential for pharmaceuticaldevelopment. In one particular embodiment, a method is provided for amechanism for targeting G3BP1 function is provided.

The current disclosure provides that knockdown of G3BP1, a stressgranule aggregating protein, increases axon growth in vitro.Introduction of the B domain of G3BP1 (which may include but is notlimited to amino acids 140-208) and may increase axon regeneration invitro and in vivo. Importantly, this may accelerate the rate ofregeneration in vivo after peripheral nerve injury. The G3BP1 B domainis highly conserved across species.

The current disclosure discloses cell permeable peptides to conservedsubregions of the G3BP1 B domain. In one embodiment, a polypeptidecomprising amino acids 190-208 of rat G3BP1 increases axonal outgrowthin cultured mammalian sensory and cortical neurons and even acceleratesaxon growth beyond the effect of injury-conditioned sensory neurons.Structure predictions of the 190-208 G3BP1 peptide show that acidicglutamate side chains of seven glutamate-proline repeats may be arrayedalong the periphery of the peptide and only conservative changes inamino acid sequence between rat, mouse and human (e.g., substitution ofaspartate for glutamate). mRNA colocalization with endogenous G3BP1protein and translation assays strongly suggest that the peptideinterferes with function of stress granules and increases intra-axonalrates of translation of proteins needed for regeneration.

The current disclosures provides a cell-permeable polypeptide thatspecifically targets mRNA storage sites in neurons and increases ratesof regeneration after traumatic injury. This may be used as the basisfor therapeutic development or could increase efficacy of existingtherapies. Additionally, there could be neuroprotective applications forneuropathic conditions (diabetic neuropathy, chemotherapy inducedneuropathy, etc.) that have much higher incidence.

FIG. 1 shows a schematic of how translation is regulated in axons.

Changes in intra-axonal translation occur after injury of peripheralnerves. mRNAs are stored before injury, and an influx of calciumrecruits mRNAs into translation. Based on localization to axons anddynamic changes after injury, it is believed that ‘stress granules’ arethe site of mRNA storage before injury and likely provide a level ofmRNA sequestration after injury/during regeneration.

FIG. 2 illustrates that immunofluorescence shows G3BP1 protein in axonsin a granular profile before injury. Seven (7) days after injury, whenaxons have fully initiated regeneration, granular profiles of G3BP1 inaxons are decreased compared to the naive nerve. There is a commensurateincrease in G3BP1 phosphorylated on serine 149. This phosphorylation isknown to ‘dissaggregate’ G3BP1.

FIG. 3 shows that consistent with phosphorylation decreasing G3BP1aggregation, phosphomimetic G3BP1 (G3BP1-S149E) shows much loweraggregation in axons than does non-phosphorylatable G3BP1 (G3BP1-S149A)and wild-type G3BP1 in axons of in cultured rat sensory neurons.

FIG. 4 illustrates differential colocalization of G3BP1 with axonalmRNAs in axons of naïve vs. injury-conditioned neurons. G3BP1colocalizes with some axonal mRNAs. Representative fluorescence in situhybridization to detect axonal mRNA combined with antibody staining forG3BP1 protein is shown on left. Quantifications of the colocalizationcoefficients are shown in the graph. This comparison is for naïve vs.injury-conditioned sensory neurons, where the conditioning injurytriggers a rapid outgrowth of axons. Importin β1 mRNA (Impβ1), encodedby an ‘injury response gene’, shows increased colocalization with G3BP1in axons of the injury-conditioned neurons. On the other hand, Neuritin1 mRNA (Nrn1), encoded by a ‘regeneration-associated gene’, showsdecreased colocalization with G3BP1 protein in axons of theinjury-conditioned neurons. GAP43 shows low colocalization coefficientsand no change with injury-conditioning.

FIG. 5 shows that expression of G3BP1 domains alters axon growth.Introduction of full length (ABCD) or separate domains of G3BP1 into DRGneurons alters axon growth in culture (schematic in upper right).Representative images of B and D domain GFP fusion proteins showlocalization into distal axons (longitudinal images in mid-left); allother fusion proteins similarly localized into axons. Representativeimages of axonal outgrowth are shown on bottom left. D domain decreasesaxon length and B domain substantially increases axon lengths.Quantification of axonal lengths over biological replicate experiments(≥3) is shown on right (*** p≤0.001, **** p≤0.0001 by a Anova with TukeyHSD post-hoc test compared to GFP control).

FIG. 6 illustrates that the G3BP1 B domain increases axon regenerationin vivo. Sciatic nerves of adult rats were transduced withadeno-associated virus 5 (AAV5) encoding GFP, G3BP1-BFP (full lengthG3BP1) or G3BP1-B domain-BFP. After seven (7) days for expression of thevirus, the animals underwent sciatic nerve crush at mid-thigh. Seven (7)days later the animals were euthanized and analyzed for axonregeneration. Representative images of immunofluorescence for axonalmarker neurofilament are shown on the left for each condition. The graphon the right shows the extent of regeneration. As FIG. 6 illustrates,regeneration is significantly enhanced by introducing the G3BP1 Bdomain.

FIG. 7 shows that the peptide for G3BP1 residues 190-208 supports axongrowth in both peripheral nervous system and central nervous systemneurons. As FIG. 7 shows, axon growth analyses for embryonic corticalneurons and naive or injury-conditioned adult dorsal root ganglion (DRG)neurons treated with cell permeable peptides corresponding to G3BP1residues 160-189 or 190-208 are shown. The peptides were designed inhouse and synthesized by Bachem. The DRG neurons were treated withpeptides by whole bath application after twelve (12) hours in cultureand analyzed twenty-four (24) hours later. These data show that the190-208 peptide can effectively increase axon growth in both naive andinjury-conditioned neurons. Cortical neurons were cultured in amicrofluidic device for three (3) days and then G3BP1 peptides were bathapplied to only the axonal compartment. As FIG. 7 illustrates, the190-208 peptide significantly increased axon growth in CNS neurons.Protein sequence alignments for corresponding regions of 190-208 peptideare shown in the upper right. The lower right shows predicted tertiarystructure of rat 190-208 peptide with approximately 180 degree verticalrotation between views. The acidic side chains of glutamates can be seenarrayed along the periphery of the structure.

Critical functions of intra-axonally synthesized proteins are thought todepend on regulated recruitment of mRNA from storage depots in axons.The current disclosure shows that axotomy induces translation of storedaxonal mRNAs via regulation of the stress granule protein G3BP1, tosupport regeneration of peripheral nerves. G3BP1 aggregates in axons ofperipheral nerves in stress granule-like structures that decrease duringregeneration, with a commensurate increase in phosphorylated G3BP1.Colocalization of G3BP1 with axonal mRNAs is also correlated with thegrowth state of the neuron. Disrupting G3BP functions by overexpressinga dominant-negative protein disassembles axonal stress granule-likestructures, activates intra-axonal mRNA translation, increases axongrowth in cultured neurons and accelerates nerve regeneration in vivo.

Injured axons in the peripheral nervous system (PNS) use locallytranslated proteins for retrograde injury-signaling and regenerativegrowth. Translation of axonal mRNAs can be activated by differentstimuli including axotomy in mature neurons and in response to guidancecues in developing neurons, indicating that a significant fraction ofaxonal mRNAs are stored until a particular stimulus activates theirtranslation. Stress granules (SG) serve as storage depots for mRNAs innon-neuronal systems, providing a mechanism to respond to cellularstress by sequestering unneeded mRNAs from translation.Aggregation-prone mutations of the SG protein TIA1 and the RNA bindingprotein TDP-43 have been shown to cause SG aggregation in neurons, butit is not known if SGs have roles in normal function of neurons.Further, although SGs have been detected in dendrites, it is not clearif functional SGs are assembled in axons. The RasGAP SH3 domain bindingprotein 1 (G3BP1) interacts with the 48S pre-initiation complex whentranslation is stalled, and it assembles SGs by virtue of its NTF2-likedomain. The current disclosure shows that translation of specific axonalmRNAs is negatively regulated in intact axons by G3BP1, but inregenerating peripheral nerves post injury, this negative regulation isremoved by dispersion of aggregated G3BP1, to then support acceleratedaxon growth. When phosphorylated on serine 149 (G3BP1^(PS149)), G3BP1'soligomerization is blocked and SGs disassemble, presumably releasingbound mRNAs for translation. Loss of G3BP1 aggregation in SG-likestructures in regenerating axons is accompanied by an increase inphosphorylated G3BP1. Disrupting G3BP1 function with a dominant-negativeapproach activates intra-axonal mRNA translation, increases axon growthin cultured neurons and accelerates nerve regeneration in vivo, andtherefore represents a new pro-regenerative therapeutic approach.

Results

Axonal G3BP1 aggregates decrease during nerve regeneration—The currentdisclosure investigated if axons of cultured primary sensory neuronscontain G3BP1 stress granule-associated protein. Sensory neurons fromadult rat dorsal root ganglia (DRG) show strong immunoreactivity forG3BP1 in cell bodies and focally along their axons, see FIG. 8A. FIG. 8Ashows Immunofluorescence for G3BP1 shows signals in cell body (asterisk)and along distal neurites (arrows) in a cultured DRG neuron. Previouswork has shown that neurites of these adult DRG neurons have axonalfeatures and lack dendritic features; we will use ‘axon’ for describingthese hereafter [scale bar=50 μm]. Axonal G3BP1 signals showed highercolocalization with known SG components than components of processingbodies (PB) that are linked to RNA degradation (FIGS. 8B and 8C). Theseaxonal G3BP1 aggregates are smaller than those described for SGs innon-neuronal cells (diameter ˜0.2-0.8 μm vs. ≥1 μm), so the axonalSG-like structures approximate the ˜250 nm diameter described for SGcore structure. FIGS. 8B and 8C show single optical planes for axons ofnaïve DRG cultures co-labeled for G3BP1, HuR, FMRP and FXR1 or G3BP1,DCP1A and XRN1 are shown as indicated; boxed region represents the areahigher magnification images are taken from (FIG. 8B). Axonal G3BP1 showshigher colocalization coefficients (FIG. 8C) for SG proteins than PBproteins (N≥30 axons over 3 repetitions) [scale bar=10 μm for largepanels and 1 μm for small panels]. PLA shows higher colocalization forG3BP1 and HuR than G3BP1 and DCP1A in axons. Confocal microscopy ofsciatic nerve sections showed robust G3BP1 signals overlappingneurofilament (NF). Using imaging parameters standardized for detectionof aggregated G3BP1, intra-axonal G3BP1 was found to be much lower inthe axons proximal to the crush site in 7 d post-injury nerves (FIG.8E). Axons are actively regenerating at 7 d after nerve crush (seeExtended Data S5B at FIG. 17B), thus, the current disclosure consideredif the decrease in axonal SG-like structures is a feature of growingaxons.

DRG neurons that are conditioned by an in vivo crush injury 7 days priorto culture show more rapid axonal outgrowth over 18-48 h in vitrocompared to uninjured (naïve) DRGs, and the rapidly growing axons ofinjury-conditioned neurons showed decreased levels of G3BP1 aggregatescompared to those of naïve DRG cultures (FIGS. 8D and 8F).

G3BP1 is phosphorylated in regenerating axons—To determine ifphosphorylation alters aggregation of axonal G3BP1, the currentdisclosure expressed non-phosphorylatable and phosphomimetic G3BP1mutants (G3BP1^(S149A). GFP and G3BP1^(S149E)-GFP, respectively) incultured DRGs. Axonal G3BP1^(S149A)-GFP showed aggregated signals thatoverlapped with HuR, while axonal G3BP1^(S149E)-GFP appeared diffuse(FIGS. 9 A and 9B). G3BP1^(S149E)GFP also showed significantly highermobility in axons than G3BP1^(S149A)-GFP, GFP, and G3BP1-GFP showedmobility intermediate between G3BP1^(S149E)-GFP and G3BP1^(S149A)-GFP(FIG. 9C, FIG. 13A). This is consistent with G3BP1^(S149A)-GFPaggregating into SG-like structures in axons. Axonal G3BP1^(PS149)immunoreactivity was increased in regenerating compared to uninjurednerves (FIGS. 9D and 9E).

Thus, axonal injury decreases the prevalence of axonal SG-likestructures, and this correlates with an increase in axonal G3BP1^(PS149)levels. Moreover, the ratio of axonal G3BP1^(PS149) to axonal G3BP1aggregates increases in distal axons and growth cones (FIGS. 9F and 9G),indicating that the axonal SG-like structures are dynamically regulatedalong the growing axon.

Axonal G3BP1 modulates axonal mRNA translation—Previous work hasdetected ribosomes and translation factors in regenerating PNS axons invivo, so the decrease in SG-like structures in distal axons couldreflect increased axonal protein synthesis. Thus, the current disclosureconsidered if axonal mRNAs colocalize with G3BP1 in cultured neurons.Endogenous Neuritin1 (Nrn1) and Importin β1 (Impβ1) mRNAs showed clearcolocalization with axonal G3BP1, but Gap43 mRNA did not (FIG. 10A). Themore rapidly growing axons of injury-conditioned DRG neurons showedhigher colocalization of Impβ1 with G3BP1 than those of naïve DRGs,while axonal Nrn1 showed the opposite (FIG. 10B). Axonal Gap43 alsoshowed overall lower G3BP1 colocalization coefficients that did notchange with injury conditioning (FIG. 10B). These distinctcolocalizations of axonal Impβ1 and Nrn1 with G3BP1 in naïve vs.injury-conditioned neurons likely reflect different functions of theencoded proteins in these different growth states.

The current disclosure moved to fluorescent reporters to determine ifaxonal SG-like structures contribute to translation. For this, thecurrent disclosure generated axonally targeted GFP^(MYR) andmCherry^(MYR) reporters containing the 5′ and 3′ untranslated regions(UTR) of Impβ1, Nrn1, and Gap43 mRNAs (GFP^(MYR)5′/3′impβ1,GFP^(MYR)5′/3′nrn1, and mCh^(MYR)5′/3′gap43, respectively; FIG. 10C).The membrane localizing myristoylation (MYR) of the fluorescent reporterproteins dramatically limits their diffusion from sites of translation,so these have provided a surrogate for localized protein synthesis indendrites and axons using fluorescence recovery after photobleaching(FRAP). The 3′ (Imp β1 and Gap43) and 5′ (Nrn1) UTRs provide axonaltargeting for reporter mRNAs, and with both 5′ and 3′UTRs, the reporterapproximates the translational regulation of the endogenous mRNAs.Recovery of axonal GFP^(MYR)5′/3′impβ1 and GFP^(MYR)5′/3′nrn1fluorescence was decreased in DRGs expressing G3BP1-BFP compared to theBFP control (FIGS. 10D, 10E and 10F), but mCh^(MYR)5′/3′gap43 recoverywas not significantly affected by G3BP1-BFP expression (FIG. 14A).Treatment with translation inhibitors confirmed that the fluorescencerecovery in axons after photobleaching represents new protein synthesis(FIGS. 10E, 10F, and FIG. 14A). Furthermore, RNA immunoprecipitation(RIP) analyses showed enrichment of GFP^(MYR)5′/3′imp/31 andGFP^(MYR)5′/3′nrn1, but not mCh^(MYR)5′/3′gap43, in G3BP1immunoprecipitates (FIG. 10G).

Acidic domain of G3BP1 increases axonal growth—G3BP1 is composed of fourseparate domains: N-terminal NTF2-like ‘A domain’, a highly acidic ‘Bdomain’, PxxP motif containing ‘C domain’, and Cterminal RNA bingingmotif containing ‘D domain’ (FIG. 11A). Expression of G3BP1 deletionconstructs in naïve DRG cultures showed that G3BP1 B, CD, BCD and Ddomain proteins all localized to axons (FIG. 17A). Neurons expressingthe G3BP1 B domain showed significantly longer axons, while thoseexpressing the D or CD domains showed shorter axons (FIG. 11B, FIG.15A). G3BP1 D domain expression decreases protein synthesis innon-neuronal cells through eIF2α phosphorylation. Interestingly,expressing G3BP1 BCD domains together reversed the growth deficit seenfor CD, pointing to a dominant effect of the B domain in absence ofG3BP1's aggregating NTF2 like region (FIG. 15A). Surprisingly,full-length G3BP1 overexpression had no significant effect on axongrowth (FIG. 15A), perhaps indicating that G3BP1 is at saturating levelsin DRG neurons. Consistent with this, siRNA-mediated knockdown (KD) ofG3BP1 significantly increased axon growth and this was completelyreversed by co-transfection with a siRNA-resistant G3BP1-GFP (FIG. 15C).However, co-transfecting with the G3BP1 B domain did not furtherincrease axon length, suggesting that B domain inhibits G3BP1 function.DRG cultures expressing full-length G3BP1-, B domain-, and CD domain-GFPshowed modest decline in neurite branching (FIG. 15A).

To determine if a smaller region of the G3BP1 B domain is sufficient toincrease axon growth, the current disclosure generatedfluorescently-labeled, cell-permeable Tat fusion peptides correspondingto residues 147-166, 168-189, and 190-208 of rat G3BP1. Peptidespenetrated the neurons in DRG cultures by 30 min. after application(FIG. 16A). When added to DRG cultures immediately after plating, boththe 147-166 and 190-208 peptides increased axon length and the 190-208peptide increased number of neurites per neuron (FIG. 16B). Effects ofthe 190-208 peptide were significantly stronger, so the currentdisclosure concentrated efforts on this peptide comparing to the 168-189peptide. To discriminate between increased axon extension vs. earlierinitiation of axon growth, the current disclosure exposed DRG culturesto peptides after the neurons had fully initiated axonal growth. Withdelayed application, the 190-208 peptide significantly increased axonlength in both naïve and pre-injured DRG neurons (FIG. 11C). Corticalneuron cultures also showed a significant increase in axon growth whenthe 190-208 peptide was applied directly to axons (FIG. 11C, FIG. 16C).

G3BP1's acidic domain accelerates PNS nerve regeneration—In light of theincreased axon growth seen above, the current disclosure asked ifintroducing the G3BP1 B domain might alter axon regeneration in vivo.For this, adult rats were transduced with adeno-associated virus (AAV)expressing B or D domains and then subjected to sciatic nerve crush 10 dlater. At 7 d after crush injury (17 d post-transduction), G3BP1-BFP,G3BP1 B domain-BFP, and G3BP1 D domain-BFP were visible in theregenerating sciatic nerve axons (FIG. 17B). The G3BP1 B domain-BFPtransduced animals showed significantly increased axon regenerationcompared to G3BP1-BFP, and G3BP1 D domain-BFP, and GFP transducedanimals (FIG. 11D, FIG. 17B).

G3BP1's acidic domain activates intra-axonal translation throughdisassembly of stress granule protein aggregates—To determine if theexogenous G3BP1 B domain interrupts function of endogenous G3BP1, thecurrent disclosure asked if expressing the B or D domains alters axonalmRNA translation. Intraaxonal translation of GFP^(MYR)5′/3′nrn1 wassignificantly higher in G3BP1 B domain expressing than in D domainexpressing DRGs, but only modestly higher GFP^(MYR)5′/3′impβ1translation was seen (FIG. 14B). Axonal mCh^(MYR)5′/3′gap43 translationshowed no significant difference between B and D domain expressingneurons (FIG. 14A). The cell-permeable G3BP1 190-208 peptide alsosignificantly rescued axonal translation of GFP^(MYR)5′/3′nrn1 mRNA inG3BP1-overexpressing DRGs, but did not affect translation ofGFP^(MYR)5′/3′impβ1 or mCh^(MYR)5′/3′gap43 mRNAs (FIG. 14C). Using apuromycinylation assay to test for translation of endogenous mRNAs,G3BP1 B domain expression led to significantly higher protein synthesisin axons but not cell bodies of cultured DRGs (FIGS. 12A, 12B, and 12C).Treatment with the cell-permeable G3BP1 190-208 peptide also increasedaxonal protein synthesis (FIG. 12D; FIG. 18A). Finally, the currentdisclosure directly tested if the interaction of endogenous mRNAs withG3BP1 is altered by B domain expression. Binding of Nrn1 and Impβ1 mRNAsto G3BP1-BFP was significantly decreased by B-domain expression, butGAP43 mRNA was not affected (FIG. 12E). Taken together, these dataindicate that the growth-accelerating G3BP1 B domain and G3BP1 190-208peptide can increase axonal protein synthesis.

Considering the effects of the G3BP1 B domain and 190-208 peptide onaxonal mRNA translation, the current disclosure reasoned that theseagents might disrupt SGs. Indeed, G3BP1 B domain expression attenuatedSG aggregation in NIH 3T3 cells exposed to sodium arsenite (FIGS. 18Band 18C), a potent inducer of SG aggregation. The current disclosurenext used time-lapse imaging with DRG cultures to determine if the Bdomain affects the axonal SG-like structures, focusing on axons beforeand after addition of the cell-permeable 190-208 peptide. The 190-208peptide caused a striking decrease in axonal G3BP1-mCh aggregates within15 min (FIG. 5f-g ). Moreover, the remaining SG-like structures in axonswere significantly smaller after 190-208 peptide treatment (FIG. 5h ),and the remaining aggregates showed greater motility than in controlconditions).

Discussion

Many studies have now documented mRNA translation in axons, and this isparticularly prominent in the PNS where intra-axonal protein synthesiscontributes to axon regeneration after injury. Some known SG proteinslocalize to axons, but intra-axonal functions for these proteins havenot been extensively tested. The current disclosure's data indicatesthat blocking G3BP1's function in assembly of axonal SG-like structuresincreases intra-axonal protein synthesis and accelerates PNS axonregeneration. Thus, axonal G3BP1 is a negative modulator of intra-axonalprotein synthesis and axon growth. With several thousand mRNAsidentified in axons of cultured neurons, it is likely that translationof numerous axonal mRNAs will be regulated by G3BP1 as the currentdisclosure shows for Impβ1 and Nrn1 mRNAs. The colocalization ofdifferent mRNAs with these G3BP1 aggregates correlates with the growthstatus of neurons, and blocking G3BP1 aggregation provides a novelstrategy to accelerate regeneration.

The difference between Impβ1 and Nrn1 colocalization with G3BP1 in naïvevs. injury conditioned neurons likely reflects different needs for thecorresponding proteins in different growth states. Nrn1 protein promotesneurite growth, and increasing axonal targeting of Nrn1 mRNA increasesaxon growth in DRG neurons. Hence, the decrease in Nrn1 mRNA associatedwith SG-like aggregates in axons of injury-conditioned neurons wouldfree the mRNA for translation to promote axon growth. On the other hand,Impβ1 mRNA translation is induced by axotomy, with its protein productproviding a retrograde signal to activate regeneration-associated geneexpression in the soma. Continued translation of Impβ1 mRNA likelydecreases axon elongation due to its role in axon length sensing.Consequently, rapid axon growth after injury conditioning would befacilitated by sequestering Impβ1 mRNA from translation.

In summary, the current disclosure points to axonal G3BP1 as a modulatorof intra-axonal protein synthesis and axon growth. Since G3BP1 isaggregated in uninjured PNS axons, the current disclosure's data pointsto unrealized functions for SG-like aggregates in axons under non-stressconditions. Preventing this SG-like aggregation of axonal proteinsduring regeneration increases the rate of axon regrowth. Consideringthat Tat fusion peptides for NR2B9c have been used in a clinical trialfor ischemic protection during endovascular repair for intracranialaneurysms, the growth-promoting effects of the cell-permeable 190-208G3BP1 peptide may represent a novel therapeutic lead for acceleratingnerve regeneration. Since peripheral nerves typically regenerate at only1-2 mm per day, accelerating axon growth rates by interfering withaxonal G3BP1 function could significantly shorten recovery times andallow axons to reach a more receptive environment to reinnervate targettissues.

FIGURE LEGENDS

FIG. 8: G3BP1 localizes to axons in stress granule-like aggregates.

FIG. 8A, Immunofluorescence for G3BP1 shows signals in cell body(asterisk) and along distal neurites (arrows) in a cultured DRG neuron.Previous work has shown that neurites of these adult DRG neurons haveaxonal features and lack dendritic features; we will use ‘axon’ fordescribing these hereafter [scale bar=50 μm]. FIGS. 8B and 8C showsingle optical planes for axons of naïve DRG cultures co-labeled forG3BP1, HuR, FMRP and FXR1 or G3BP1, DCP1A and XRN1 are shown asindicated; boxed region represents the area higher magnification imagesare taken from (b). Axonal G3BP1 shows higher colocalizationcoefficients (c) for SG proteins than PB proteins (N≥30 axons over 3repetitions) [scale bar=10 μm for large panels and 1 μm for smallpanels].

FIG. 9: G3BP1 is phosphorylated in regenerating axons.

FIG. 9A shows axons of DRG neurons transfected with indicated G3BP1constructs vs. eGFP are shown. G3BP1-GFP and G3BP1^(S149A)-GFP showprominent aggregates in axons that colocalize with HuR (arrows). Incontrast, axonal signals for G3BP1^(S149E)-GFP and eGFP appear morediffuse [Scale bar=5 μm]. FIG. 9B shows quantification of axonalaggregates for G3BP1-GFP, G3BP1^(S149A)-GFP, and G3BP1^(S149E)-GFP isshown as average±SEM (N≥10 neurons over 3 repetitions; * p≤0.005 byone-way ANOVA with Tukey HSD post-hoc).

FIG. 9C shows FRAP analyses for neurons transfected with constructs asin A are shown as average normalized % recovery±SEM (see FIG. 13A forrepresentative FRAP image sequences). G3BP1^(S149A)-GFP shows much lowerrecovery than G3BP1^(S149E)-GFP; G3BP1-GFP is intermediate betweenG3BP1^(S149A)-GFP and G3BP1^(S149E)-GFP (N≥13 axons over 3repetitions; * p≤0.05 between G3BP1^(S149A). GFP vs. G3BP1^(S149E)GFP byone-way ANOVA with Tukey HSD post-hoc). Only the 0-320 sec recoverysignals for GFP are shown (at 840 sec GFP showed 85.5±4.7% recovery;p≤0.0001 vs. G3BP1^(S149E)-GFP by one-way ANOVA with Tukey HSDpost-hoc).

FIGS. 9D and 9E shows exposure-matched confocal images for G3BP1^(PS149)and NF are shown for sciatic nerve (d) as in FIG. 8D. There is astriking increase in G3BP1^(PS149) signal in the regenerating axons.Quantification of these signals (e) shown as mean±SEM (N=3; * p≤0.05 byone-way ANOVA with Tukey HSD post-hoc) [scale bar=20 μm].

FIGS. 9F and 9G, shows distal axons of cultured DRGs immunostained withpan-G3BP1 vs. G3BP1^(PD149) antibodies are shown as indicated (f).Aggregates of G3BP1 are visible in the axon shaft (arrow), but decreasemoving distally towards the growth cone (arrowhead). G3BP1^(PS149)signals are fairly consistent and extend into the growth cone(arrowhead). Quantification of signal (g) shows significant increase inratio of G3BP1^(PS149) immunoreactivity to G3BP1 aggregates movingdistally to the growth cone (N≥9 neurons each over 3 repetitions; *p≤0.05 vs. 70-80 μm bin by one-way ANOVA with Tukey HSD post-hoc) [Scalebar=20 μm].

FIG. 10: G3BP1 regulates translation of axonal mRNAs.

FIG. 10A shows images of FISH/IF for indicated mRNAs and G3BP1 proteinare shown for axons of naïve and 7 d injury-conditioned DRG neurons.Colocalization panel represents the mRNA:G3BP1 colocalization in asingle optical plane [Scale bar=5 μm].

FIG. 10B shows quantification of colocalizations for Nrn1, Impβ1, andGap43 mRNAs with G3BP1 in axons of neurons cultured from naïve or 7 dinjury-conditioned animals shown as average Pearson's coefficient±SEM(N≥21 neurons over 3 repetitions; ** p≤0.01 and *** p≤0.005 by one-wayANOVA with Tukey HSD posthoc).

FIG. 10C shows schematics of translation reporter constructs used inpanels d-f.

FIG. 10D shows representative FRAP image sequences for DRG neuronsco-transfected with GFP^(MYR)5′/3′nrn1 plus BFP or G3BP1-BFP. Boxedregions represent the photobleached ROIs.

FIGS. 10E and 10F show quantifications of FRAP assays from DRGsexpressing GFP^(MYR)5′/3′nrn1 (e) or GFP^(MYR)5′/3′impβ1 (f) translationreporters along with G3BP1-BFP or control BFP are shown as normalized,average % recovery±SEM (see FIG. 14A for mCh^(MYR)5′/3′gap43; N≥11neurons over 3 repetitions; * p≤0.05, and ** p≤0.01 by one-way ANOVAwith Tukey HSD post-hoc). Cultures treated with translational inhibitorsshowed reduced fluorescence recovery that paralleledG3BP1-overexpressing neurons.

FIG. 10G shows HEK293T cells transfected with GFP^(MYR)5′/3′nrn1,GFP^(MYR)5′/3′ impβ1, and mCh^(MYR)5′/3′gap43 show significantenrichment of GFP^(MYR)5′/3′nrn1 and GFP^(MYR)5′/3′ impβ1 mRNAscoimmunoprecipitating with G3BP1 immunoprecipitate vs. control. Westernblot validating G3BP1 immunoprecipitation shown as inset. Values shownas average percent bound mRNA relative to input SEM (N=4 culturepreparations; * p≤0.05 by Student's t-test).

FIG. 11: G3BP1 acidic domain expression accelerates nerve regeneration.

FIG. 11A shows a schematic of G3BP1 domains as defined by Tourriere etal. (2003).

FIG. 11B shows representative images for NF-labeled DRG neuronstransfected with indicated constructs. Images were acquired at 60 hpost-transfection [scale bar=100 μm]. FIG. 17A shows axonal localizationof these G3BP1-GFP domain proteins and FIG. 15A shows quantitation ofaxon growth from G3BP1 domain-expressing DRG neurons.

FIG. 11C shows quantitation of axon growth from DRGs (left) and corticalneurons (right) treated with cell-permeable 168-189 or 190-208 G3BP1peptides. For DRGs, peptides were added to dissociated naïve or 7 dinjury-conditioned DRGs at 12 h and axon growth was assessed at 36 h invitro. For cortical neurons, peptides were added to the axonalcompartment of microfluidic devices at 3 d in vitro (DIV), and axongrowth was assessed at 6 DIV. See FIG. 16C for images of corticalcultures (N≥95 over 3 DRG cultures and 9 microfluidic devices over 3cultures; *** p≤0.005 by one-way ANOVA with Tukey HSD post-hoc).

FIG. 11D shows the extent of axon regeneration at 7 day post sciaticnerve crush in adult rats transduced with AAV5 encoding G3BP1-BFP, G3BP1B domain-BFP, G3BP1 D domain-BFP, or GFP control as mean axonal profilesrelative to crush site (0 mm)±SEM. For representative NF stained imagessee Extended Data S5B (N≥5 animals per condition; *=p≤0.05 and =p≤0.0001by one-way ANOVA with Tukey HSD post-hoc).

FIG. 12: G3BP1 acidic domain increases axonal mRNA translation anddisassembles stress granules.

FIGS. 12A, 12B, and 12C show representative images for puromycin (Puro)incorporation in DRG neurons transfected with indicated constructs areshown (a). Quantitation shows no change in cell body Puro signals (b),while the axons show a significant increase in Puro signals in the G3BP1B domain expressing neurons (c) (N≥100 axons and N≥30 cell bodies over 3repetitions; **p≤0.01 by one-way ANOVA with Tukey HSD posthoc) [scalebar=5 μm].

FIG. 12D show puro incorporation in DRG axons was also significantlyincreased by the 190-208 G3BP1 peptide treatment compared to control and168-189 peptide exposure (N≥83 axons over 3 DRG cultures; ****p≤0.0001by one-way ANOVA with Tukey HSD post-hoc). Representative images areshown in FIG. 18A.

FIG. 12E shows RTddPCR for axonal mRNAs associated with G3BP1-GFP in DRGneurons as average % mRNA associated with G3BP1-GFP±SEM. Nrn1 and Imp81mRNAs association with G3BP1-GFP significantly reduces by expression ofB domain (N=4 culture preparations; * p≤0.05, ** p≤0.01 by Student'st-test).

FIG. 12F show representative images of G3BP1-mCh in DRG axons treatmentwith 190-208 G3BP1 peptide for 15 min are shown. Axon tracing wasgenerated from DIC images [scale bar=10 μm].

FIG. 12G show density of G3BP1-mCh aggregates along 100 μm length axonsfrom DRG cultures treated as in FIG. 12F are shown (N≥51 axons over 3repetitions; **** p≤0.0001 by Student's t-test).

FIG. 12H size of G3BP1-mCh aggregate is shown as indicated bins for fromDRG cultures treated as in FIG. 5f (N≥50 aggregates over 3 repetitions;**** p≤0.0001 for entire population distributions by Kolmogorov-Smirnovtest).

METHODS

Animal use and survival surgery—Institutional Animal Care and UseCommittees of University of South Carolina, Emory University, andWeizmann Institute of Science approved all animal procedures. MaleSprague Dawley rats (175-250 g) were used for all sciatic nerve injuryand DRG culture experiments. Embryonic day 18 (E18; male and female) ratpups were used for cortical neuron culture experiments. Isofluorane wasused for anesthesia for AAV transduction and peripheral nerve injury.

For peripheral nerve injury, anesthetized rats were subjected to asciatic nerve crush at midthigh as described. In cases where animalswere transduced with virus prior to injury, AAV5 was injected into theproximal sciatic nerve 7 d prior to crush injury (at sciatic notchlevel; 9-14×10¹⁰ particles in 0.6 M NaCl).

Axoplasm was obtained from sciatic nerve at 3, 7, 14, 21 and 28 d aftercrush injury at mid-thigh level. 2 cm segments of nerve proximal to theinjury site (or equivalent level on contralateral [naïve] side) weredissected and axoplasm extruded into 20 mM HEPES [pH 7.3], 110 mMpotassium acetate, and 5 mM magnesium acetate (nuclear transport buffer)supplemented with protease/phosphatase inhibitor cocktail (Roche) andRNasin Plus (Promega). After clearing by 20,000×g centrifugation at 4°C. for 30 min, supernatants were mixed with 3 volumes of Trizol LS(Invitrogen) for protein precipitation. 3 animals were used for eachtime point.

Cell culture—For primary neuronal cultures, L4-5 DRG were harvested inHybernate-A medium (BrainBits) and then dissociated as known to those ofskill in the art. After centrifugation and washing in DMEM/F12 (LifeTechnologies), cells were resuspended in DMEM/F12, 1×N1 supplement(Sigma), 10% fetal bovine serum (Hyclone), and 10 μM cytosinearabinoside (Sigma). Dissociated DRGs were plated immediately onlaminin/poly-L-lysine-coated coverslips or transfected (see below) andthen plated on coated coverslips.

For cortical neuron cultures, E18 cortices were dissected in Hibernate E(BrainBits) and dissociated using the Neural Tissue Dissociation kit(Miltenyi Biotec). For this, minced cortices were incubated in apre-warmed enzyme mix at 37° C. for 15 min; tissues were then trituratedand applied to a 40 μm cell strainer. After washing and centrifugation,neurons were seeded at a density of 1×105 cells per poly-D-lysine-coatedmicrofluidic device (Xona Microfluidics). NbActive-1 medium (BrainBits)supplemented with 100 U/ml of Penicillin-Streptomycin (LifeTechnologies), 2 mM L-Glutamine (Life Technologies), and 1×N21supplement (R&D Systems) was used as culture medium.

NIH-3T3 and HEK293T cells were maintained in DMEM (Life Technologies)supplemented with 10% FBS (Gibco) and 100 U/ml ofPenicillin-Streptomycin (Life Technologies).

For DRG neuron transfection, dissociated ganglia were pelleted bycentrifugation at 100×g for 5 min and resuspended in ‘nucleofectorsolution’ (Rat Neuron Nucleofector kit; Lonza). 5-7 μg plasmid waselectroporated using an AMAXA Nucleofector apparatus (program SCN-8;Lonza). For siRNA transfection, 100 nM siRNAs (Dharmacon) were used withDharmaFECT 3 reagent and incubated for 36 h. G3BP1 siRNA sequence: 5′ccacauaggagcugggaauuu 3′. Non-targeting siRNAs (siCon) were as control.RTddPCR was used to test the efficiency of depletion (see below).HEK293T cells were transfected using Lipofectamine® 2000 permanufacturer's instructions (Invitrogen). AAV5 preparations weretitrated in DRG cultures by incubating with 1.8-2.8×1010 particles ofAAV5 overnight.

For arsenic treatment to induce SG aggregation, transfected NIH3T3 cellswere grown to 60-80% confluence and were treated with 0.5 mM sodiumarsenite (Sigma) for 30 min.

For peptide treatments, 10 μM Tat-fused peptides were added todissociated DRG cultures at 2 or 12 h after plating. Neurite outgrowthwas assessed 24 h after addition of peptides. For the cortical cultures,10 μM peptide was applied to the axonal compartment at 3 d in vitro(DIV) and axonal growth was assessed at 6 DIV.

Plasmid and viral expression constructs—All fluorescent reporterconstructs for analyses of RNA translation were based on eGFP withmyristoylation element (GFP^(MYR); originally provided by Dr. ErinSchuman, Max-Plank Inst., Frankfurt) or mCherry plasmid withmyristoylation element (mChMT). Reporter constructs containing 5′ and3′UTRs of rat Nrn1 and Gap43 mRNAs have been published. For Impβ1, the5′UTR was cloned by PCR and inserted directly upstream of the initiationcodon in GFP^(MYR)3′impβ1 (includes 3′UTR of rat Impβ1 mRNA).

Human G3BP1 wild type, S149A, S149E and deletion constructs asGFP-tagged proteins were generously provided by Dr. Jamal Tazi, Institutde Génétique Moléculaire de Montpellier. The G3BP1-mCherry construct wasgenerated by PCR, amplifying G3BP1 CDS with 5′ NheI and 3′ HindIIIrestriction sites. After NheI+HindIII digestion, G3BP1 CDS was subclonedinto NheI+HindIII-digested pmCherry-N1 vector (Clontech).

AAV5 preparations were generated in UNC Chapel Hill Viral Vector Core.All plasmid inserts were fully sequenced prior to generating AAV.BglII+XhoI digested human G3BP1 cDNA (from pGFPG3BP1) was subcloned intoBamHI+XhoI digested pAAV-cDNA6-V5His vector (Vector Biolabs). G3BP1deletion constructs were amplified by PCR with terminal HindIII and Xholrestriction sites (primer sequences available on request). Afterdigestion with HindIII and Xhol, products were cloned intoHindIII+Xhol-digested pAAV-cDNA6-V5His vector. BFP was excised from thepTagBFP-N vector (Evrogen) using EcoRI+NotI and ligated in-framedirectly 3′ of the G3BP1 sequences in pAAV-cDNA6-V5His.

Generation of Tat-tagged G3BP1 B domain peptides—Three peptides weregenerated from the rat G3BP1 B domain sequence (amino acids 140-220;UniProt ID # D3ZYS7_RAT) by Bachem Americas, Inc. Peptides weresynthesized with N-terminal dansyl chloride or FITC and N- or C-terminalHIV Tat peptide for cell permeability; the Tat sequence was placed atthe least conserved end of the sequence based on P-BLAST of sequencesavailable in UniProt database. Peptide sequences with Tat sequence were(Tat shown in italics): 147-166, SEQ ID NO: 13 SEQ ID NO: 13 Glu Glu SerGlu Glu Glu Val Glu Glu Pro Glu Glu Arg Gln Gln Ser Pro Glu Val Val TvrGlv Asn Lvs Lvs Asn Asn Gln Asn Asn Asn; 168-189, SEQ ID NO: 14 Am AspSer Gly Thr Phe Tyr Asp Gln Thr Val Ser Asn Asn Leu Glu Glu His Leu GluGlu Pro Tvr Glv Asn Lvs Lvs Asn Asn Gln Asn Asn Asn; and 190-208, SEQ IDNO: 15 Tvr Glv Asn Lvs Lvs Asn Asn Asn Gln Asn Asn Asn Val Val Glu ProGlu Pro Glu Pro Glu Pro Glu Pro Glu Pro Glu Pro Val Ser.

Immunofluorescent staining—All procedures were performed at roomtemperature (RT) unless specified otherwise. Cultured neurons were fixedin 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) andprocessed as described. Primary antibodies consisted of: rabbitanti-G3BP1 (1:200, Sigma), RT97 mouse anti-neurofilament (NF; 1:500,Devel. Studies Hybridoma Bank), and rabbit anti-G3BP1^(PS149) (1:300,Sigma). FITC-conjugated donkey anti-rabbit and Cy3-conjugated donkeyanti-mouse (both at 1:200, Jackson ImmunoRes.) were used as secondaryantibodies.

For G3BP1 colocalization with SG and PB proteins, Zenon antibodylabeling kit (Life Technologies) was used to directly label antibodieswith fluorophores. Combinations of rabbit anti-G3BP1 (Sigma)+Alexa-488,rabbit anti-HuR (Millipore)+Alexa-405, rabbit anti-FMRP (Cell SignalingTech)+Alexa-555, and rabbit anti-FXR1 (kind gift from Dr. Khandiah,Institut Universitaire en Sant4 Mentale de Quebec)+Alexa-633 or rabbitanti-G3BP1+Alexa-488, rabbit anti-DCP1A (Abcam)+Alexa-405, and rabbitanti-XRN1 (Bethyl Lab)+Alexa-633 were used at 1:50 dilution for eachantibody. Equal amounts of rabbit-IgG labeled with Alexa-405, -488, -555and -633 were used as control.

For quantifying axonal content of G3BP1 and G3BP1^(PS149) in peripheralnerve, sciatic nerve segments were fixed for 4 h in 4% PFA and thencryoprotected overnight in 30% sucrose, PBS at 4° C. 10 μm cryostatsections were processed for immunostaining as previously described.Primary antibodies consisted of rabbit anti-G3BP1 (1:100), rabbitanti-phospho-G3BP1PS149 (1:100), and RT97 mouse anti-NF (1:300).Secondary antibodies were FITC-conjugated donkey anti-rabbit andCy3-conjugated donkey anti-mouse (both at 1:200, Jackson ImmunoRes.).Immunoblotting confirmed the specificity of the anti-G3BP1 and-G3BP1PS149 antibodies.

Paraffin sections were used for analyses of nerve regeneration. Forthis, 10 μm thick paraffin sections of sciatic nerve were deparaffinizedin 100% xylene (2×10 min) followed by 100% ethanol (2×10 min). Sectionswere rehydrated by sequential incubations in 95, 75 and 50% ethanol for5 min each, and then rinsed in deionized water. Sections werepermeabilized in 0.3% Triton X-100 in PBS, and then rinsed in PBS for 20min and equilibrated in 50 mM Tris [pH 7.4], 150 mM NaCl, 1% heat-shockbovine serum albumin (BSA), and 1% protease-free BSA (Roche) (‘IFbuffer’). Sections were then blocked in IF buffer plus 2% heat-shockBSA, and 2% fetal bovine serum for 1.5-2 h. After blocking, samples wereincubated overnight at 4° C. in humidified chamber with the primaryantibodies in IF buffer. Samples were washed in IF buffer three timesand then incubated in secondary antibodies diluted in IF buffer for 45min. Samples were washed in IF buffer three times followed by rinse inPBS and deionized water. Primary antibodies consisted of: RT97 mouseanti-NF (1:300) and rabbit anti-RFP (1:100, Rockland Immun. Chem.). TheRFP antibody was confirmed to detect BFP by immunoblotting (see below)and immunolabeling of transfected DRG neurons (data not shown).Secondary antibodies were used as above.

All samples were mounted with Prolong Gold Antifade (Invitrogen) andanalyzed by epifluorescent or confocal microscopy. Leica DMI6000epifluorescent microscope with ORCA Flash ER CCD camera (Hamamatsu) orLeica SP8X confocal microscope with HyD detectors was used for imagingunless specified otherwise. For quantitation between samples, imagingparameters were matched for exposure, gain, offset and post-processing.

Fluorescence in-situ hybridization (FISH)—For FISH, DRG cultures werefixed for 15 min in 2% PFA in PBS. RNA-protein colocalization wasperformed using custom 5′ Cy3-labeled ‘Stellaris’ probes (probesequences available upon request; Biosearch Tech.). Scrambled probeswere used as control for specificity; samples processed without additionof primary antibody were used as control for antibody specificity.Primary antibodies consisted of rabbit anti-G3BP1 (1:100) and RT97 mouseanti-NF (1:200). FITC-conjugated donkey anti-rabbit and Cy5-conjugateddonkey anti-mouse (both at 1:200) were used as secondaries. Samples weremounted as above and analyzed using a Leica SP8X confocal microscope.Samples were post-processed with Huygens deconvolution integrated intothe Leica LSM software and analyzed as outlined below for RNA-proteincolocalization.

Fluorescence recovery after photobleaching (FRAP)—FRAP was used to testfor axonal mRNA protein synthesis using diffusion-limited GFP^(MYR) andmCherry^(MYR) reporters as described with minor modifications. In eachcase, DRG neurons were co-transfected withGFP^(MYR)5′/3′nrn1+mCherry^(MYR)5′/3′gap43 orGFP^(MYR)5′/3′impβ1+mCherry^(MYR)5′/3′gap43 so that recovery of bothreporters could be analyzed simultaneously. Cells were maintained at 37°C., 5% CO2 during imaging sequences. 488 nm and 514 nm laser lines onLeica SP8X confocal microscope were used to bleach GFP and mCherrysignals, respectively (Argon laser at 70% power, pulsed every 0.82 secfor 80 frames). Pinhole was set to 3 Airy units to ensure full thicknessbleaching and acquisition (63×/1.4 NA oil immersion objective). Prior tophotobleaching, neurons were imaged every 60 sec for 2 min to acquirebaseline fluorescence the region of interest (ROI; 15% laser power,498-530 nm for GFP and 565-597 nm for mCherry emissions, respectively).The same excitation and emission parameters were used to assess recoveryover 15 min post-bleach with images acquired at 30 sec intervals. Todetermine if fluorescence recovery in axons was from translation,cultures were treated with 150 μg/ml cycloheximide (Sigma) or 100 μManisomycin (Sigma) for 30 min. prior to photobleaching forGFP^(MYR)5′/3′nrn1+mCherry^(MYR)5′/3′gap43 andGFP^(MYR)5′/3′impβ1+mCh^(MYR)5′/3′gap43 transfected DRGs, respectively.For peptide treatments, G3BP1-mCh transfected DRG neurons were treatedwith 10 μM G3BP1 peptides after acquiring the baseline expressionvalues. Photobleaching followed by analyses of recovery was performedafter 30 min of peptide exposure.

For testing G3BP1 protein mobility in axons, DRG neurons weretransfected with G3BP1^(S149A)-GFP or G3BP1^(S149E)-GFP and imaged asabove but only the 488 nm laser was used for photobleaching (Argon laserat 70% power, pulsed every 0.82 sec for 80 frames).

Fluorescent intensities in the ROIs were calculated by the Leica LASXsoftware. For normalizing across experiments, fluorescence intensityvalue at t=0 min post-bleach from each image sequence was set as 0%. Thepercentage of fluorescence recovery at each time point afterphotobleaching was then calculated by normalizing relative to thepre-bleach fluorescence intensity (set at 100%).

Live cell imaging for G3BP1-mCherry granules—DRG neurons weretransfected with G3BP1-mCherry, and 36 h later distal axons were imagedusing Leica SP8X confocal microscope with environmental chambermaintained at 37° C., 5% CO2 (with 63×/1.4 NA oil immersion objective).Confocal pinhole was set to 0.43 airy units. G3BP1-mCherry signals wereimaged in axon shaft every 2 sec for 100 frames (at 540 nm excitationand 23% white light laser power; 565-597 nm emission). To study theeffect of the G3BP1 190-208 peptide on the G3BP1-mCherry granules, 10 μMFITC-conjugated peptide was added to the media and 15 min later imagingwas continued.

For G3BP1-mCherry aggregates, a 100 μm of the axon shaft was considered(≥200 μm from cell body). Thresholding was applied to acquired imagesequences using ImageJ to generate binary masks. ImageJ particleanalyzer was used for analysis. G3BP1 aggregates with area of ≥1 μm2were considered as SG-like structures. For analyzing the G3BP1 aggregatevelocity, ImageJ Trackmate plug-in was used.

Puromycinylation assay—To visualize newly synthesized proteins incultured neurons, the current disclosure used the Click-iT® Plus OPPProtein Synthesis Assay Kit per manufacturer's instructions(Invitrogen/Life Technologies). Briefly, 3 DIV cultures were incubatedwith 20 μM O-propargyl-puromycin (OPP) for 30 min at 37° C. OPP-labeledproteins were detected by crosslinking with Alexa Fluor-594 picolylazide molecule. Coverslips were then mounted with Prolong Gold Antifade(Invitrogen) and imaged with Leica DMI6000 epifluorescent microscope asabove. ImageJ was used to quantify the Puromycinylation signals indistal axons and cell bodies.

Immunoblotting—For immunoblotting, protein lysates or immunoprecipitateswere denatured by boiling in Laemmle sample buffer, fractionated bySDS-PAGE, and transferred to nitrocellulose membranes. Blots wereblocked for 1 h at room temperature with 5% non-fat dry milk inTris-buffered saline with 0.1% Tween 20 (TBST) for anti-GFP, -tagBFP,and -G3BP1 antibodies; 5% BSA in TBST was used for blockinganti-G3BP1^(PS149) antibody. Primary antibodies diluted in appropriateblocking buffer were added to the membranes and incubated overnightincubation at 4° C. with rocking. Primary antibodies consisted of:rabbit anti-G3BP1 (1:2,000; Sigma), rabbit anti-G3BP1PS149 (1:1,000;Sigma), rabbit anti-GFP (1:5,000; Abcam), rabbit anti-TagBFP (1:4,000;Evrogen). After washing in TBST, blots were incubated HRP-conjugatedanti-rabbit IgG antibodies (1:5,000; Jackson lab) diluted in blockingbuffer for 1 h at room temperature. After washing signals were detectedusing ECL Prime™ (GE Healthcare).

RNA immunoprecipitation (RIP)—HEK293T cells or DRG neurons were lysed in100 mM KCl, 5 mM MgCl², 10 mM HEPES [pH 7.4], 1 mM DTT, and 0.5% NP-40(RIP buffer) supplemented with 1×protease inhibitor cocktail (Roche) andRNasin Plus (Invitrogen). Cells were passed through 25 Ga needle 5-7times and cleared by centrifugation at 12,000×g for 20 min. Clearedlysates were pre-absorbed with Protein A-Dynabeads (Invitrogen) for 30min. Supernatants were then incubated with primary antibodies for 3 hand then immunocomplexes precipitated with Protein G-Dynabeads(Invitrogen) for additional 2 h at 4° C. with rotation. Mouse anti-G3BP1(5 μg, BD Biosciences) and rabbit anti-GFP (5 μg, Abcam) antibodies wereused for immunoprecipitation. Beads were washed 6 times with cold RIPbuffer. Bound RNAs were purified and analyzed by RTddPCR (see below).

RNA isolation and PCR analyses—RNA was isolated from immunoprecipitatesand cultures using the RNeasy Microisolation kit (Qiagen). Fluorimetrywith Ribogreen (Invitrogen) was used for RNA quantification. Foranalyses of total RNA levels and inputs for RIP analyses, RNA yieldswere normalized across samples prior to reverse transcription usingSensifast (Bioline). For RIP assays, an equal proportion of each RIP wasused for reverse transcription with Sensifast. ddPCR products weredetected using Evagreen or Tagman primer and probe sets (Biorad orIntegrated DNA Tech; sequences available on request) and QX200™ dropletreader (Biorad). In GFP RIP experiment, B domain-BFP expressionconsistently increased G3BP1-GFP levels in the DRG neurons. So the levelof mRNA precipitating with G3BP1-GFP was normalized to the G3BP1-GFPsignals from immunoblotting across each sample in each experiment.

Image analyses and processing—For protein-protein and protein-mRNAcolocalization, xyz scan sequence captured 100 μm segments of axon shaft(separated from cell body and growth cone by ≥200 μm) were deconvolvedusing Huygens Hyvolution software. Colocalization was analyzed usingImageJ JACoP plug-in(https://imagej.nih.gov/ij/plugins/track/jacop.html) to calculatePearson's coefficient. These coefficient calculations were independentlyvalidated with Volocity software (Perkin Elmer).

For analyses of protein levels in tissues, z planes of the xyz tilescans from 3-5 locations along each nerve section were analyzed usingImageJ. Colocalization plug-in was used to extract protein signals thatoverlap with axonal marker (NF) in each plane, with the extracted‘axon-only’ signal projected as a separate channel. For calculatingaxonal G3BP1 aggregate and G3BP1^(PS149) signal intensities, absolutesignal intensity was quantified in each xy plane of the ‘Colocalization’extracted images for axonal only G3BP1 and G3BP1^(PS149) using ImageJ.Protein signal intensities across the individual xy planes were thennormalized to NF immunoreactivity area. The relative protein signalintensity was averaged for all image locations in each biologicalreplicate.

For neurite outgrowth, images from 60 h DRG cultures were analyzed forneurite outgrowth using WIS-Neuromath. Axon morphology was visualizedusing GFP and/or NF immunofluorescence as described.

To assess regeneration in vivo, tile scans of NF-stained nerve sectionswere post-processed by Straighten plug-in for ImageJ(http://imagej.nih.gov/ij/). NF positive axon profiles were then countedin 30 μm bins at 0.3 mm intervals distal from crush site. Number of axonprofiles present in the proximal crush site was treated as the baseline,and values from the distal bins were normalized to this to calculate thepercentage of regenerating axons.

Statistical analyses—Kaleidagraph (Synergy) or Prism (GraphPad) softwarepackages were used for statistical analyses. One-way ANOVA was used tocompare means of independent groups and Student's t-test was used tocompare smaller sample sizes of the in vivo analyses. p values of ≤0.05were considered as statistically significant.

In one embodiment, see FIG. 19, a method 100 for treating nerve injuryin a mammal may be provided. At step 102, a polypeptide comprisingbetween 15 and 20 amino acids may be introduced to a nerve injury sitein the mammal. At step 104, the polypeptide may interfere with functionof stress granules and increase intra-axonal rates of translation ofproteins needed for nerve regeneration. The polypeptide may have anamino acid sequence set as forth in SEQ ID NO: 2. At step 106, thepolypeptide may specifically target mRNA storage sites in neurons andincreases rates of neuron regeneration. At step 108, the polypeptide maydisrupt G3BP functions. At step 110, disruption of G3BP functions may:activate, intra-axonal mRNA translation; increase axon growth inneurons; and accelerate nerve regeneration in vivo. Disruption of G3BPfunctions may be accomplished via siRNA-mediated knockdown of G3BP1. Atstep 112, disrupting G3BP1's function in an assembly of axonal stressgranule structures may increases intra-axonal protein synthesis andaccelerates peripheral nervous system axon regeneration. Acceleratedaxon growth regeneration may be facilitated by sequestering Imp81 mRNAfrom translation.

FIG. 20 shows an alternative method 200 wherein. At step 202, G3BPfunction may be disrupted. At step 204, a dominant-negative protein maybe overexpressed. At step 206, the dominant-negative protein maydisassemble axonal stress granule-like structures, activate intra-axonalmRNA translation, increase axon growth in neurons; and accelerate nerveregeneration in vivo. The protein may comprise between 15 and 20 aminoacids and may have an amino acid sequence as set forth in SEQ ID NO: 2.At step 208, the protein is cell permeable and targets mRNA storagesites in neurons. At step 210, disruption of G3BP functions may beaccomplished via siRNA-mediated knockdown of G3BP1. At step 212,disrupting G3BP1's function in an assembly of axonal stress granulestructures may increase intra-axonal protein synthesis and accelerateperipheral nervous system axon regeneration. At step 214, preventingstress granule-like aggregation of axonal proteins during regenerationmay increase the rate of axon regrowth.

While the present subject matter has been described in detail withrespect to specific exemplary embodiments and methods thereof, it willbe appreciated that those skilled in the art, upon attaining anunderstanding of the foregoing may readily produce alterations to,variations of, and equivalents to such embodiments. Accordingly, thescope of the present disclosure is by way of example rather than by wayof limitation, and the subject disclosure does not preclude inclusion ofsuch modifications, variations and/or additions to the present subjectmatter as would be readily apparent to one of ordinary skill in the artusing the teachings disclosed herein.

What is claimed is:
 1. A prophylactic method for accelerating nerverecovery treating nerve injury in a mammal comprising: introducing apolypeptide, wherein the polypeptide is amino acid sequence SEQ ID NO:2, comprising between 15 and 20 amino acids to increase axon growth inneurons or accelerating nerve regeneration at a nerve injury site in themammal; and wherein the polypeptide interferes with function of stressgranules and increases intra-axonal rates of translation of proteinsneeded for nerve regeneration.
 2. The method of claim 1, wherein thepolypeptide specifically targets mRNA storage sites in neurons andincreases rates of neuron regeneration.
 3. The method of claim 1,wherein the polypeptide disrupts G3BP functions.
 4. The method of claim3, wherein disruption of G3BP functions: activates intra-axonal mRNAtranslation; increases axon growth in neurons; and accelerates nerveregeneration in vivo.
 5. The method of claim 3, wherein disruption ofG3BP functions is accomplished via transfection of siRNA to promotesiRNA-mediated knockdown of G3BP1.
 6. The method of claim 5, whereindisrupting G3BP1's function in an assembly of axonal stress granulestructures increases intra-axonal protein synthesis and acceleratesperipheral nervous system axon regeneration.
 7. The method of claim 6,wherein accelerated axon growth regeneration is facilitated bysequestering Imp61 mRNA from translation.
 8. A method of increasing axongrowth in neurons or accelerating nerve regeneration disrupting G3BPfunctions comprising: overexpressing a dominant-negative protein,wherein the protein is introduced by a transporter molecule; wherein thedominant-negative protein: disassembles axonal stress granule-likestructures activates intra-axonal mRNA translation; increases axongrowth in neurons; and accelerates nerve regeneration in vivo; andwherein the protein is amino acid sequence SEQ ID NO:
 2. 9. The methodof claim 8, wherein the protein is cell permeable and targets mRNAstorage sites in neurons.
 10. The method of claim 8, wherein preventingstress granule-like aggregation of axonal proteins during regenerationincreases the rate of axon regrowth.